Process for producing ethanol by using recombinant coryneform bacterium

ABSTRACT

The present invention provides a process for highly efficiently producing ethanol at a high productivity consisting of using a coryneform bacterium which has been transformed by a DNA containing a gene expressing pyruvate decarboxylase activity and, if desired, a gene expressing alcohol dehydrogenase activity under a regulatory sequence allowing for the expression, under ethanol production conditions wherein this bacterium does not substantially proliferate to produce ethanol.

FIELD OF THE INVENTION

[0001] The present invention relates to a process for producing ethanol.Particularly, the present invention relates to a process of producingethanol wherein sugars such as glucose are used as raw material, andmore particularly, a process for highly efficiently producing ethanol ata high productivity consisting of using a coryneform bacterium which hasbeen transformed by a DNA containing gene expressing pyruvatedecarboxylase (referred to hereinafter as PDC) activity and, if desired,a gene expressing alcohol dehydrogenase(referred to hereinafter as ADH)activity under a regulatory sequence allowing the expression, andfermenting a raw material, sugars such as glucose, under ethanolproduction conditions wherein this bacterium does not substantiallyproliferate to produce ethanol, which is then collected.

BACKGROUND ART

[0002] Up to now, ethanol has been prepared by chemical synthesis viaethylene from fossil resources such as coal and petroleum, or afermentation of sugars from biomass resource such as plant by amicroorganism such as yeast or bacteria. Among these, a process forproducing ethanol by fermentation using a renewable biomass resource inlight of energy resource or environmental problems has been noticed.

[0003] A process for producing ethanol by a conventional large-scaleindustrial fermentation is a process by fermenting starch or sugars fromvarious biomass resources, namely, a technology based on a brewage forpotable ethanol. However, the use of a yeast as a fermentationmicroorganism results in slow rate of ethanol production and alsodifficulties such as complicated fermentation control because of thenecessity of aeration, even though the fermentation is performed underanaerobic condition.

[0004] It is well known to use a strain belonging to Zymomonas as afermentation microorganism (Japanese Patent Publication No. H7-59187).

[0005] It is recognized that a fermentation process by using Zymomonasleads to increased rate of ethanol production compare to that by using ayeast. Improvement of ethanol production efficiency by biotechnologicalmodification of various microorganism species has been proposed(Japanese Patent Publication Nos. H5-502366, H6-504436, and H6-505875).

[0006] In the above technologies, 2 genes from Zymomonas mobilis, oneencoding for PDC activity (which catalyses the conversion of pyruvicacid in the glycolytic pathway to acetoaldehyde) and another ADHactivity (which catalyses the conversion of acetoaldehyde to ethanol)were inserted into host (enteric bacteria such as Escherichia coli orErwinia chrysanthe) under a regulatory sequence allowing for theirexpression. Consequently, ethanol fermentation with high productivitywas achieved by using such transformants.

[0007] According to the abovementioned technologies, improvement inethanol productivity owes to the high growth and high cell density ofthe transformants in the fermentor. Such fermentation involving cellgrowth presents many inefficiencies including low conversion efficiencyof sugar material to ethanol because the sugar material is used as anenergy supply source for growth of said microorganism, low ethanolproduction rate during the period until the microorganism reachesstationary growth phase and complications in fermentor control due tothe change of density of the microorganism accompanying the cell growththereof.

[0008] Another technological problem is separation of toxic substanceswhen Escherichia coli used as a host cell. Escherichia coli issucceptible to bacteriolysis leading to possible contamination offermentation products with toxic intracellular protein.

[0009] From this point of view, improvement of host microorganism to betransformed is desirable.

[0010] Problem to be Solved by the Invention

[0011] The present invention provides a new process for producingethanol by using biomass resources as raw material for sugars, whereinbetter excellent technology for efficiently producing ethanol at a highproductivity has been accomplished and wherein various problems pointedout in the foregoing prior art have been solved.

[0012] Means to Solve the Problem

[0013] The present inventors have made extended studies to solve theproblems mentioned above, and have found that efficient production ofethanol at a high productivity can be achieved by using a coryneformbacterium which has been transformed with a gene expressing PDCactivity, and if desired, a gene expressing ADH activity under aregulatory sequence allowing for expression, and then, performingfermentation under ethanol production conditions wherein the transformedcoryneform bacterium does not substantially proliferate, whereby thepresent invention has been accomplished.

[0014] One of the characteristics of the present invention is that ahost bacterium to be transformed is a coryneform bacterium and thatethanol is prepared under conditions wherein the transformed coryneformbacterium does not substantially proliferate.

[0015] Embodiment of the Invention

[0016] A gene expressing ADH activity and a gene expressing PDC activityfrom various microorganisms can be used in the present invention.Although each of the genes may be derived from different mcroorganisms,Zymomonas mobilis can be preferably used.

[0017] Specifically, the following gene, which have been already clonedand sequenced, can be used.

[0018] Genes Expressing ADH Activity

[0019] Origin Zymomonas mobilis:

[0020] ADHI gene (acc.NO.M32100) (J.Bacteriol.vol. 172, 2491-2492(1990))

[0021] ADHII gene (acc.NO.X17065,M15394) (J.Bacteriol.vol.169,2591-2597(1987))

[0022] Origin Saccharomyces cerevisiae:

[0023] ADH1 gene (J. Biol. Chem.vol.257,3018-3025(1982))

[0024] ADH2 gene (J. Biol. Chem.vol.258,2674-2682(1983))

[0025] ADH3 gene (Nature,vol.387,90-93(1997))

[0026] ADH4 gene (acc.NO.X05992) (Mol.Gen.Genet.vol. 209,374-381 (1987))

[0027] ADH5 gene (EMBO J.vol.13,5795-5809(1994))

[0028] Origin Sinorhizobium meliloti:

[0029] ADH gene (Biochim.Biophys.Acta vol.1384,197-203 (1998))

[0030] Origin Salmonella typhimurium:

[0031] ADH gene (J.Bacteriol.vol.181(17),5317-5329(1999)

[0032] Origin Mycobacterium tuberculosis:

[0033] ADH gene (Nature,vol.393,537-544(1998))

[0034] Origin Esherichia coli:

[0035] ADH gene (DNA Res.vol.3,137-155(1996))

[0036] Genes Expressing PDC Activity

[0037] Origin Zymomonas mobilis:

[0038] PDC gene (J.Bacteriol.vol.170,3310-3313(1988))

[0039] Origin Saccharomyces cerevisiae:

[0040] PDC1 (acc.NO.X77316) (Nucleic Acids Res.voll4, 893-63-8977(1986))

[0041] PDC6 (acc.NO.X66843,X55905) (J.Bacteriol.vol. 173, 7963-7969(1991))

[0042] PDC5 (acc.NO.X15668)

[0043] PDC2 (acc.No.X65608) (Mol.Gen.Genet.vol241,657-666(1993))

[0044] Origin Bacillus subtilis:

[0045] pdhA gene/pdhB gene (acc.NO.AF012285) (J.Bacteriol. vol.172,5052-5063(1990))

[0046] Origin Thiobacillus ferrooxidans:

[0047] pdhA gene/pdhB gene (acc.NO.U81808)(Microbiology, vol.142,2543-2548(1996)).

[0048] It is necessary for the two genes to be under a regulatorysequence in order to express their activity, although they are notnecessarily required to be under a common regulatory sequence. They maybe under separate regulatory sequences and in some cases on a differentplasmids and at a different sites on a chromosome.

[0049] “Under a regulatory sequence” as used herein means that anintended gene can autonomously replicate by, for example,collaboratively acting with a promoter, inducer, operator, ribosomebinding site and transcription terminator etc.

[0050] A host microorganism transformed according to the presentinvention is a coryneform bacterium. The coryneform bacterium is aGram-positive bacterium and different from a Gram-negative bacteriumsuch as E. coli which is used as a host bacterium in the aforementionedprior art. It is noteworthy that USP5,482,846 and USP5,916,787 discloseda process for transforming a Gram-positive bacterium by a geneexpressing ADH activity and PDC activity. However, the Gram-positivebacteria as used therein are Bacillus, Lactobacillus; Fibribacter,Ruminococcus, Pediococcus, Cytophaga, Cellulomonas, Bacteroides,Clostridium, Bacillus subtilis, and Bacillus polymyxa, but it is nottaken into account that a coryneform bacterium can be used as the hostcell to be transformed.

[0051] One of the major characteristics of the present invention is thata coryneform bacterium, which does not originally have a function ofethanol production from pyruvic acid, is selected as the host to betransformed. Use of coryneform bacterium transformed according to thepresent invention, enabled us to prepare ethanol under conditions inwhich no substantial cell growth was observed, in contrast to the usualprocess for producing ethanol by fermentation with yeast, Zymomonas orenteric bacteria such as E. coli transformed according to theaforementioned prior art.

[0052] Thus, by producing ethanol without cell growth, manytechnological problems resulting from cell growth as described above,are overcome. This has a significant value as a practical industrialproduction technology. The present inventors have discovered that such atechnology can be achieved by using a coryneform bacterium.

[0053] Substantial suppression of growth of coryneform bacterium can beachieved by subjecting the aerobic bacterium to anaerobic conditions, orrestricting the essential nutritional requirement, biotin (J.Industrial. Microbiol. vol.5,289-294.(1990)). In addition,biotechnological regulation of a function of a gene regulating growthwould be an effective means.

[0054] Although ethanol fermentation under anaerobic condition hasalready been accomplished, the technology emphasizes or is accompaniedby cell growth. On the contrary, the present invention is quitedifferent to the extent that the present invention is intended tosuppress the substantial growth of a coryneform bacterium in an ethanolreaction vessel.

[0055] The aforementioned prior art discloses that both anaerobic andaerobic conditions lead to an equivalent growth of a transformant andproductivity of ethanol, which differs from the present invention intechnological idea or content.

[0056] An essential element of the present invention is to introduceinto a coryneform bacterium a DNA containing a gene expressing PDCactivity and, if required, a gene expressing ADH activity under aregulatory sequence allowing for expression.

[0057] In this process, it is essential to introduce a gene expressingPDC activity, but not essential to introduce a gene expressing ADHactivity. This is because usually a coryneform bacterium is deemed tohave neither genes expressing the foregoing respective enzymes but it isuncertain regarding ADH. Thus, it cannot be denied that certaincoryneform bacteria have the gene expressing ADH enzyme activity. Inthis case, it is basically enough to use only a gene expressing PDCenzyme activity as the gene to be introduced.

[0058] However, both genes are preferably introduced to effect highertransformation even if the coryneform bacterium used as a hostoriginally has an ability to produce ADH.

[0059] There is no limitation as the type of aerobic coryneform bacteriatransformed with the gene expressing PDC activity and, if desired, thegene expressing ADH activity, as long as it is a coryneform bacteriumwhich is able to growth under usual aerobic conditions. Examples thereofare Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, andMicrococcus. And more particularly, examples of Corynebacterium areCorynebacterium glutamicum ATCC13032, ATCC13058, ATCC13059, ATCC13060,ATCC13232, ATCC13286, ATCC13287, ATCC13655, ATCC13745, ATCC13746,ATCC13761, ATCC14020, and ATCC31831. Examples of Brevibacterium areBrevibacterium lactofermentum ATCC13869, Brevibacterium flavumMJ-233(FERM BP-1497) and MJ-233AB-41 (FERM BP-1498), Brevibacteriumammoniagenes ATCC6872. Examples of Arthrobacter are. Arthrobacterglobiformis ATCC8010, ATCC4336, ATCC21056, ATCC31250, ATCC31738 andATCC35698.

[0060] As a transformation, there are processes for recombining achromosome of a host bacterium with both genes, or a process to use arecombinant plasmid wherein both genes have been incorporated into aplasmid which can autonomously replicate in a host bacterium.

[0061] A plasmid vector used for such purposes may be one containing agene with an autonomous replication function in a coryneform bacterium.As specific example, pAM330(Agric. Biol. Chem.vol. 48,2901-2903 (1984)and Nucleic Acids Symp Ser.Vol.16,265-267 (1985)) (from Brevibacteriumlactofermentum 225), pHM1519(Agric. Biol. Chem.vol.48, 2901-2903(1984)(from Corynebacterium glutamicum ATCC13058), pCRY30(Appl. Environ.Microbiol.vol. 57, 759-764(1991)), pEKO, pEC5, pEKEx1 (Gene vol.102,93-98 (1991)), and pCG4 (J. Bacteriol. Vol.159, 306-311 (1984))(fromCorynebacterium gluatmicum T250).

[0062] Construction of a plasmid used for transformation of coryneformbacteria in the present invention can be effected, for example, in thecase of a gene from Zymomonas mobilis being used, by linking aregulatory sequence such as an appropriate promoter and terminator withDral-Dral 1.4 kb gene fragment containing an intact ADH gene (J.Bacteriol.vol.169,2591-2597(1987)), and Dral-Dral 1.8 kb containing anintact PDC gene (J. Bacteriol. vol.169,949-954(1987)) respectively,which is then inserted at an appropriate restriction site of any one ofa plasmid vector as exemplified above.

[0063] An example of a promoter to express ADH gene and PDC gene in theaforementioned recombinant plasmid is, but not limited to, the one whichis originally carried by a coryneform bacterium. It may be any basesequence which has a function to initiate the transcription of ADH geneand PDC gene. An example of a terminator which is put in the downstreamof ADH gene and PDC gene under a regulatory sequence is, but not limitedto, the one which is originally carried by a coryneform bacterium. Itmay be, for example, any base sequence having a function to terminatethe transcription of ADH gene and PDC gene, such as the terminator fortryptophan operon from E. coli.

[0064] In the present invention, cultivation of the coryneformbacterium, which is performed prior to introduction of a plasmid vectorcontaining such as the intended gene into the coryneform bacterium, canbe performed under usual aerobic conditions. A medium used forcultivation of usual microorganism can be used as culture medium. Forexample, to a general medium containing natural nutrients such as meatextract, yeast extract, and peptone etc., solution of inorganic saltsuch as ammonium sulfate, potassium phosphate, and magnesium sulfate isadded, if necessary.

[0065] A process for introducing a plasmid vector containing theintended gene after the foregoing cultivation includes, but is notlimited to, electroporation and the CaCl₂ method as long as such processenables introduction of a gene into a coryneform bacterium. In anembodiment thereof, for example a known method can be used for anelectrical pulsation (Agric.Biol. Chem.vol.54, 443-447 (1990),Res.Microbiol.vol.144,181-185(1993)).

[0066] As for the introduction of the intended gene into a chromosome, asimilar method is available, for example, technology such as thatdescribed in DNA sequence vol.3,303-310 (1993) is available.

[0067] A method for selecting the transformed coryneform bacteriaemploys antibiotic resistance, whereby a gene encoding for the saidresistance is introduced into a plasmid vector or a chromosomecontaining the intended gene, and then plating the transformedcoryneform bacterium, onto a plate containing an appropriateconcentration of the said antibiotic. As an embodiment thereof, forexample a method as described in Agric. Biol. Chem. vol.54,443-447(1990), Res. Microbiol. vol. 144, 181-185 (1993) is available.

[0068] Basically, in order to use an aerobic coryneform bacterium forthe process of preparing ethanol of the present invention, a largeamount of transformed coryneform bacteria must be cultured under usualaerobic conditions first. The present cultivation can be performed in asimilar manner as a cultivation of a coryneform bacterium prior totransformation.

[0069] Transformed coryneform bacteria of the present invention whichwere cultured under aerobic conditions are harvested by centrifugation,membrane filtration or chemically treated (for example, immobilizationwith carrageenan) in order to be used for a subsequent reaction toproduce ethanol.

[0070] For the reaction to produce ethanol, an aqueous solutioncontaining the appropriate inorganic salt or buffer is preferably used.To the aqueous solution, sugars, such as glucose which is material forethanol production, are added.

[0071] The reaction to produce ethanol is performed under conditionswherein growth of the transformed aerobic coryneform bacterium issubstantially suppressed. Various methods, as described above, can beemployed to substantially suppress the growth, and it is adequate to putthe transformed aerobic coryneform bacterium under anaerobic condition.A system for reaction to produce ethanol may be batchwise or continuousreaction, and preferably continuous in view of achieving highproductivity. Use of continuous reaction system in the process of thepresent invention does not lead to substantial growth of the coryneformbacterium and therefore the operation control thereof is easier thanthat of a conventional method, which is accompanied by cell growth,

[0072] Sugars used as raw material for ethanol production include,preferably, but are not limited to, glucose which leads to rapid ethanolproduction.

[0073] “Anaerobic conditions” as used herein means any conditionsresulting in lowered concentration of dissolved oxygen in aqueoussolution, provided that a trace amount of the dissolved oxygen should beallowed as long as growth of the transformed coryneform bacterium issubstantially inhibited. This condition is achieved by, for example,carrying out the reaction without aeration in a sealed container, orperforming the reaction while supplying an inert gas such as nitrogen.The temperature for ethanol production is usually 15° C.-45° C., andpreferably 25° C.-37° C. pH during the reaction is adjusted to the rangeof 5-9, and preferably 7-8. The coryneform bacterium used for ethanolproduction in the present invention can be used at a very highconcentration such as 1 g/l-1500 g/l because it is not substantiallygrowing during the reaction.

[0074] In ethanol production of the present invention, the cells are notsubstantially growing during the reaction, and raw material sugars arenot consumed as a source of nutrition for growth. In addition, no needfor induction period such as the exponential growth phase for providinga high-density culture of bacteria means that ethanol production withhigh density (high concentration) of cells from the early stage ofreaction is possible, thereby highly efficient ethanol production at ahigh productivity can be achieved.

[0075] Ethanol prepared according to the process as stated above isisolated from the reaction and purified, if necessary, and utilized asfuel ethanol or industrial raw chemical material.

[0076] The following examples illustrate the present invention indetail, but should not be deemed to limit the scope of the invention.

EXAMPLES Example 1

[0077] Cloning of a DNA Containing ADH Gene-and PDC Gene Fragments fromZymbmonas mobilis ATCC29191.

[0078] (A) Extraction of Total DNA from Zymomonas mobilis ATCC29191:

[0079] To 1L of a growth medium [Composition:20 g of Glucose, 5 g ofYeast Extract and 1000 ml of distilled water], a platinum loopful ofZymomonas mobilis was inoculated. It was then anaerobically cultured at30° C. till at late exponential growth phase, and harvested.

[0080] Bacterial cell obtained were suspended at the concentration of 10mg/ml to 15 ml of a solution containing 10 mg/ml lysozyme, 10 mM NaCl,20 mM Tris buffer(pH 8.0) and 1 mM EDTA-2Na(the concentration of eachcomponents shows the final concentration). Then, protease K was added atthe final concentration of 100 μg/ml, and heated at 37° C. for 1 hour.In addition, sodium dodecyl sulfate (SDS) was added to the finalconcentration of 0.5%, and kept warm to 50° C. for 6 hours to lead tobacteriolysis. To this bacteriolyzed solution, an equal amount ofphenol/chloroform solution was added, gradually shaken at roomtemperature for 10 min, and then the total volume was centrifuged(5,000×g, 20 min,10-12° C.) to separate a fraction of supernatant. Tothis supernatant, sodium acetate was added to become at a concentrationof 0.3 M, and then, double volume of ethanol was gradually added. DNAexisting between a water layer and ethanol layer was taken up by glassrod, washed with 70% ethanol and then air-dried. To the DNA obtained, 5ml of a solution of 10 mM Tris buffer (pH 7.5)-1 mM EDTA-2Na was addedand allowed to stand over night at 4° C. followed by used for thesubsequent experiments.

[0081] (B) Cloning of a DNA Fragment Containing ADH Gene and PDC Genefrom Zymomonas mobilis:

[0082] PCR was conducted by using chromosomal DNA prepared in Example1(A) as a template. In order to clone ADH gene and PDC gene In the PCR,the following each one pair of primers is synthesized by using “394DNA/RNA synthesizer” (Applied Biosystems) and used.

[0083] ADH Gene-amplifying Primer

[0084] (a-1) 5′—tct cga gct ctg tag ggt gag gtt ata gct—3′ (SEQ ID NO:1)

[0085] (b-1) 5′—ctc tgg tac ctc aag aca gga cgg aaa acc—3′ (SEQ ID NO:2)

[0086] Primer (a-1) contains SacI restriction site and primer b-1)contains KpnI restriction site.

[0087] PDC Gene-amplifying Primer

[0088] (a-2) 5′—tct cga att ctt gaa tat atg gag taa gca—3′(SEQ ID NO: 3)

[0089] (b-2) 5′—tct cga gct caa act aga gga gct tgt taa—3′(SEQ ID NO: 4)

[0090] Primer (a-2) contains EcoRI restriction site and primer (b-2)contains SacI restriction site.

[0091] Actually, PCR was conducted under the following conditions byusing “DNA thermal cycler” (Perkin Elmer Cetus Co., Ltd.) and Taq DNAPolymerase/TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) as a reaction reagent.Reaction: (10×)PCR buffer 10 μl 1.25 mM dNTP mixture 16 μl Template DNA10 μl (Content of DNA: less than 1 μM) Two primers as describedabove^(*)) 1 μl (respectively) (Final concentration: 0.25 μM)Recombinant Taq DNA Polymeraze 0.5 μl Sterilized water 61.5 μl

[0092] PCR Cycle Denaturation process: 94° C. 60 sec Annealing process:52° C. 60 sec Extension process: 72° C. 120 sec 

[0093] One cycle consisting of the above processes was repeated thirtycycles.

[0094] Ten microliter of reaction prepared as stated above was subjectedto an electrophoresis with 0.8% agalose gel, and about 1.2 kb of a DNAfragment was detected for ADH gene, and about 1.7 kb of a DNA fragmentfor PDC gene.

Example-2

[0095] Preparation of a Recombinant Coryneform bacterium by ADH Gene andPDC Gene from Zymomonas mobilis.

[0096] (A) Construction of Shuttle Vector:

[0097] Five microliter of about 3.0 kb HindIII-HpaI DNA fragmentcontaining ORF1(rep)of plasmid pAM330 inherent to Brevibacteriumlactofermentum ATCC13869 (Yamaguchi,R. et al., Agric. Biol. Chem. 50,2771-2778 (1986), Japanese Patent Publication No. S58-67679) and 2 μl ofplasmid pHSG398 (Takara Shuzo Co., Ltd.) which was cleaved with arestriction enzyme HindIII were mixed, to which each component of 1 μlof T4 DNA ligase 10× buffer, 1 unit of T4 DNA ligase was added, made 10μl with sterilized distilled water, and reacted at 15° C. for 3 hours tocombine.

[0098] By using plasmid mixture thus obtained and calcium chloridemethod [Journal of Molecular Biology, 53, 159(1970) ], Escherichia coliJM109(Takara Shuzo Co., Ltd.) was transformed, and was plated ontomedium (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 16 g ofagar were dissolved in 1 L of distilled water) containing 50 mg ofchloramphenicol.

[0099] A strain grown on the medium was cultured in a liquid medium in ausual manner, plasmid DNA was extracted from the culture medium, and theplasmid was cleaved with a restriction enzyme to confirm the insertedfragment. As a result, in addition to 2.2 kb of DNA fragment of plasmidpHSG398 about 3.0 kb of the inserted DNA fragment was identified.

[0100] This Escherichia coli-coryneform bacterium shuttle vector isreferred to as pKP1.

[0101] (B) Ligation of Tac Promoter with ADH Gene and PDC Gene:

[0102] Five microliter of about 1.2 kb DNA fragment containing ADH genefrom Zymomonas mobilis amplified in Example 1(B), and 2 μl of plasmidpTrc99A(Pharmacia) containing tac promoter were respectively cleavedwith restriction enzymes SacI and KpnI, the restriction enzymes wereinactivated by treating at 70° C. for 10 min, and then both arecombined, to which each component consisting of 1 μl of T4 DNA ligase10× buffer and 1 unit of T4 DNA ligase was added, made 10 μl withsterilized distilled water, and reacted at 15° C. for 3 hours to bind.This is used as ligation solution A.

[0103] Similarly, 5 μl of about 1.7 kb DNA fragment containing PDC genefrom Zymomonas mobilis amplified in Example 1(B) and 2 μl of plasmidpTrc99A(Pharmacia) containing tac promoter were respectively cleavedwith restriction enzymes EcoRI and SacI, and the restriction enzymeswere inactivated by treating at 70° C. for 10 min and then both arecombined, to which each component consisting of 1 μl of T4 DNA ligase10× buffer and 1 unit of T4 DNA ligase was added, made 10 μl withsterilized distilled water, and reacted at 15° C. for 3 hours to ligate.This is used as ligation solution B.

[0104] By using each of two ligation solutions A and B, and calciumchloride method [Journal of Molecular Biology, 53, 159(1970)],Escherichia coli JM109 (Takara Shuzo Co., Ltd.) was respectivelytransformed and was spread on a medium (10 g of tryptone, 5 g of yeastextract, 5 g of NaCl and 16 g of agar were dissolved in 1 L of distilledwater) containing 50 mg of ampicillin.

[0105] Each of strains grown on the media was cultured in a liquidmedium, plasmid DNA was extracted from the culture medium, the plasmidwas cleaved with restriction enzyme (SacI, KpnI) for ligation solutionA, and with restriction enzyme (EcoRI, SacI) for ligation solution Brespectively, and the inserted fragment was confirmed. As a result, inaddition to about 4.2 kb of DNA fragment from plasmid pTrc99A, aninserted DNA fragment with 1.2 kb in length was identified for ADH gene(ligation solution A), and an inserted fragment with 1.7 kb in lengthfor PDC gene (ligation solution B).

[0106] A plasmid containing ADH gene is referred to as pTrc99A-ADH, anda plasmid containing PDC gene is referred to as pTrc99A-PDC.

[0107] Then, in order to prepare a DNA fragment wherein ADH gene waslinked to tac promoter and a DNA fragment wherein PDC gene was linked totac promoter by using PCR method in which plasmid pTrc99A-ADH andplasmid pTrc99A-PDC were used as a template, the following each one pairof primers was synthesized by using “394 DNA/RNA synthesizer” (AppliedBiosystems).

[0108] (a-3)5′—ctc tag atc tcc gac atc ata acg gtt ctg—3′ (SEQ ID NO: 5)

[0109] (b-3)5′—ctt ctc tca tcc gcc aaa ca—3′ (SEQ ID NO: 6)

[0110] In addition, primer (a-3) contains BgIII restriction site.

[0111] Actually, PCR was conducted under the following conditions byusing “DNA thermal cycler” (Perkin Elmer Cetus Co., Ltd.) and Taq DNAPolymerase/TaKaRa Ex Taq)(Takara Shuzo Co., Ltd.) as a reaction reagent.Reaction: (10×)PCR buffer 10 μl 1.25 mM dNTP mixture 16 μl TemplateDNA^(*)) 10 μl (Content of DNA: less than 1 μM) Primers (a-3) and (b-3 )as described above 1 μl (respectively) (Final concentration: 0.25 μM)Recombinant Taq DNA Polymerase 0.5 μl Sterilized water 61.5 μl

[0112] PCR Cycle Denaturation: 94° C. 60 sec Annealing: 52° C. 60 secExtension: 72° C. 120 sec 

[0113] One cycle consisting of the above processes was repeated thirtycycles.

[0114] Ten microliter of reaction prepared as stated above was subjectedto electrophoresis on 0.8% agalose gel, and about 1.4 kb of DNA fragmentwas detected for tac promoter-linked ADH gene, and about 1.9 kb of DNAfragment for tac promoter-linked PDC gene.

[0115] (C) Insertion of a DNA Fragment in which ADH Gene is Linked toTac Promoter, and a DNA Fragment in which PDC Gene is Linked to TacPromoter into a Shuttle Vector:

[0116] Five μl of reaction of about 1.9 kb of DNA fragment, wherein PDCgene was linked to tac promoter, of which amplified product wasconfirmed in the above Example 2(B) was completely cleaved withrestriction enzymes BgIII and BamHI, and 2 μl of plasmid pKP1 preparedin the above Example 2 (A) was completely cleaved with restrictionenzyme BamHI respectively. After the restriction enzymes wereinactivated by treating at 70° C. for 10 min, both are combined, towhich each component consisting of 1 μl of T4 DNA ligase 10× buffer and1 unit of T4 DNA ligase was added, made 10 μl with sterilized distilledwater, and reacted at 15° C. for 3 hours to bind.

[0117] By using the obtained plasmid mixture and calcium chloride method[Journal of Molecular Biology, 53, 159(1970)], Escherichia coliJM109(Takara Shuzo Co., Ltd.) was transformed, and was spread on amedium (tryptone:10 g, yeast extract:5 g, NaCl:5 g and agar:16 g weredissolved in 1 L of distilled water) containing 50 mg ofchloramphenicol.

[0118] A strain grown on this media was cultured in a liquid medium,plasmid DNA was extracted from the culture medium, the plasmid wascleaved with restriction enzyme, and the inserted fragment wasconfirmed. As a result, in addition to about 5.2 kb of DNA fragment fromplasmid pKP1 prepared in the above Example 2(A), an inserted DNAfragment with about 1.9 kb in length was identified. This plasmid isreferred to as pKP1-PDC. This plasmid has only one BamHI restrictionsite.

[0119] Five μl of reaction of about 1.4 kb of DNA fragment, wherein ADHgene was linked to tac promoter, of which amplified product wasconfirmed was completely cleaved with restriction enzymes BgIII andBamHI, and 2 μl of the above plasmid pKP1-PDC was completely cleavedwith restriction enzyme BamHI respectively. After the restrictionenzymes were inactivated by treating at 70° C. for 10 min, both arecombined, to which each component consisting of 1 μl of T4 DNA ligase10× buffer and 1 unit of T4 DNA ligase was added, made 10 μl withsterilized distilled water, and reacted at 15° C. for 3 hours to bind.

[0120] By using the obtained plasmid mixture and calcium chloride method[Journal of Molecular Biology, 53, 159(1970)], Escherichia coliJM109(Takara Shuzo Co., Ltd.) was transformed and was spread on a medium(tryptone:10 g, yeast extract:5 g, NaCl:5 g and agar:16 g were dissolvedin 1 L of distilled water) containing 50 mg of chloramphenicol.

[0121] A strain grown on this media was cultured in a liquid medium,plasmid DNA was extracted from the culture medium, the plasmid wascleaved with restriction enzyme, and then, the inserted fragment wasconfirmed. As a result, in addition to about 7.1 kb of DNA fragment fromplasmid pKP1 prepared in the above Example 2(A), an inserted DNAfragment with about 1.4 kb in length was identified.

[0122] This plasmid was named pKP1-ADH (SEQ ID NO: 7).

[0123] (D) Transformation of Coryneform Bacterium:

[0124] The plasmid was introduced into Corynebacterium glutamicumATCC13032 by using electrical pulse method (Y. Kurusu, et al., Agric.Biol. Chem. 54: 443-447. 1990 and A. A. Vertes, et al., Res. Microbiol.144:181-185. 1993). A transformant thus obtained, Corynebacteriumglutamicum pKP1-PDC-ADH/13032 was deposited with National Institute ofBioscience and Human Technology (1-3 Higashi 1-Chome, Tsukuba-shi,Ibaraki-ken, Japan) on Jun. 6, 2000 (accession No. FERM P-17887), andthen transferred to the International Deposit under Budapest Treaty onMay 31, 2001 (International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology, AIST TsukubaCentral 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566,Japan, accession No. FERM BP-7621).

Example 3

[0125] Ethanol Production by Corynebacterium glutamicum ATCC13032Transformant (Corynebacterium glutamicum pKP1-PDC-ADH/13032)

[0126] A medium consisting of urea:40 g, (NH₄)₂SO₄:140 g, KH₂PO₄:5.0 g,K₂HPO₄:5.0 g, MgSO₄.7H₂O:5.0 g, FeSO₄.7H₂O:200 mg, MnSO₄.nH₂O:200 mg,D-biotin:2000 μg, thiamine hydrochloride: 1000 μg, yeast extract: 10 g,casamino acids:10 g and distilled water:10 l(pH 6.6) was poured in 500ml portion into 2 l Erlenmeyer flask, sterilized at 120° C. for 15 min,to which 40 ml of an sterilized aqueous solution of 50% glucose wasadded. To the medium, Corynebacterium glutamicum strain which wastransformed by introducing the above pKP1-PDC-ADHplasmid(pKP1-PDC-ADH/13032)was inoculated and cultured at 33° C. for 24hours with stirring (aerobic culture). After completion of culture, itwas centrifuged (8000 g, 20 min) to herveste the bacterial cells. Thetotal volume of the obtained bacterial cells were subjected to thefollowing reactions.

[0127] Five hundred ml of reaction consisting of (NH₄)₂SO₄:23 g,KH₂PO₄:0.5 g, K₂HPO₄:0.5 g, MgSO₄.7H₂O:0.5 g,FeSO₄.7H₂O:20 mg,MnSO₄.nH₂O:20 mg, D-biotin:200 μg, thiamine hydrochloride:100 μg, sodiumcarbonate 20 g, distilled water:1000 ml was poured into 1 L jarfermentor and the above bacterial cells and 50% glucose solution 120 mlwere added, which was reacted at 30° C. with slowly stirring (200 rpm)under sealed condition (anaerobic reaction). After 2 and 4 hours, therespective reactions were centrifuged (8000 rpm, 15 min, at 4° C.), andeach of supernatants thus obtained was then subjected to gaschromatography to be revealed the ethanol production at theconcentration of 3.79 and 6.96(g ethanol/l), respectively.

[0128] These results are shown in the following Table 1. TABLE 1Reaction time(hr) 2 4 Concentration of 3.79 6.96 produced ethanol (gethanol/l) Mean rate of 1.89 1.74 ethanol production (g ethanol/l/hr)

Example 4

[0129] Ethanol Production by using Brevibacterium lactofermentumATCC13869 Transformant (pKP1-PDC-ADH/13869)

[0130] Plasmid pKP1-PDC-ADH prepared according to the method of Example2 (C) was introduced into Brevibacterium lactofermentum ATCC13869 in asimilar manner as the method of Example 2 (D). The obtainedtransformant, Brevibacterium lactofermentum pKP1-PDC-ADH/13869 wasdeposited with National Institute of Bioscience and Human Technology(1-3 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan) on Jun. 6, 2000(accession No. FERM P-17888), and then transferred to the InternationalDeposit under Budapest Treaty on May 31, 2001 (International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-Chome,Tsukuba-shi, Ibaraki-ken, 305-8566, Japan, accession No. FERM BP-7622).

[0131] In a similar manner as the method of Example 3, the obtainedcoryneform bacterium transformant was aerobically cultured, andsubjected to a reaction to anaerobically produce ethanol. After 4 hours,the concentration of ethanol in a reactor was 4.21(g ethanol/l). Thus,the mean rate of ethanol production is 1.05(g ethanol/l/hr).

[0132] Effect of the Invention

[0133] According to the present invention, reaction of an aerobiccoryneform bacterium transformed with ADH gene and PDC gene underanaerobic condition allows efficiently producing ethanol at a highproductivity.

[0134] Sequence Listing Free Text

[0135] Seq Id No: 1:

[0136] A primer for amplifying ADH gene.

[0137] Seq Id No: 2:

[0138] A primer for amplifying ADH gene.

[0139] Seq Id No: 3:

[0140] A primer for amplifying PDC gene.

[0141] Seq Id No: 4:

[0142] A primer for amplifying PDC gene.

[0143] Seq Id No: 5:

[0144] A primer for amplifying ADH gene linking with tac promoter.

[0145] Seq Id No: 6:

[0146] A primer for amplifying PDC gene linking with tac promoter.

[0147] Seq Id No: 7:

[0148]E. coli/coryneform bacteria shuttle vector having PDC gene linkingwith tac promoter ADH gene linking with tac promoter.

1 7 1 30 DNA Artificial Sequence A primer for amplifying ADH gene 1tctcgagctc tgtagggtga ggttatagct 30 2 30 DNA Artificial Sequence Aprimer for amplifying ADH gene 2 ctctggtacc tcaagacagg acggaaaacc 30 330 DNA Artificial Sequence A primer for amplifying PDC gene 3 tctcgaattcttgaatatat ggagtaagca 30 4 30 DNA Artificial Sequence A primer foramplifying PDC gene 4 tctcgagctc aaactagagg agcttgttaa 30 5 30 DNAArtificial Sequence A primer for amplifying ADH gene linking with tacpromoter 5 ctctagatct ccgacatcat aacggttctg 30 6 20 DNA ArtificialSequence A primer for amplifying PDC gene linking with tac promoter 6cttctctcat ccgccaaaca 20 7 8500 DNA Artificial SequenceE.coli/coryne-form bacteria shuttle vector having PDC gene linking withtac promoter ADH gene linking with tac promoter 7 aagcttactg gccgtcgttttacaacgtcg tgactgggaa aaccctggcg ttacccaact 60 taatcgcctt gcagcacatccccctttcgc cagctggcgt aatagcgaag aggcccgcac 120 cgatcgccct tcccaacagttgcgcagcct gaatggcgaa tgagcttctt ccgcttcctc 180 gctcactgac tcgctgcgctcggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 240 ggcggtaata cggttatccacagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 300 aggccagcaa aaggccaggaaccgtaaaaa ggccgcgttg ctggcgtttt tccataggct 360 ccgcccccct gacgagcatcacaaaaatcg acgctcaagt cagaggtggc gaaacccgac 420 aggactataa agataccaggcgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 480 gaccctgccg cttaccggatacctgtccgc ctttctccct tcgggaagcg tggcgctttc 540 tcaatgctca cgctgtaggtatctcagttc ggtgtaggtc gttcgctcca agctgggctg 600 tgtgcacgaa ccccccgttcagcccgaccg ctgcgcctta tccggtaact atcgtcttga 660 gtccaacccg gtaagacacgacttatcgcc actggcagca gccactggta acaggattag 720 cagagcgagg tatgtaggcggtgctacaga gttcttgaag tggtggccta actacggcta 780 cactagaagg acagtatttggtatctgcgc tctgctgaag ccagttacct tcggaaaaag 840 agttggtagc tcttgatccggcaaacaaac caccgctggt agcggtggtt tttttgtttg 900 caagcagcag attacgcgcagaaaaaaagg atctcaagaa gatcctttga tcttttctac 960 ggggtctgac gctcagtggaactccgtcga acggaagatc acttcgcaga ataaataaat 1020 cctggtgtcc ctgttgataccgggaagccc tgggccaact tttggcgaaa atgagacgtt 1080 gatcggcacg taagaggttccaactttcac cataatgaaa taagatcact accgggcgta 1140 ttttttgagt tatcgagattttcaggagct aaggaagcta aaatggagaa aaaaatcact 1200 ggatatacca ccgttgatatatcccaatgg catcgtaaag aacattttga ggcatttcag 1260 tcagttgctc aatgtacctataaccagacc gttcagctgg atattacggc ctttttaaag 1320 accgtaaaga aaaataagcacaagttttat ccggccttta ttcacattct tgcccgcctg 1380 atgaatgctc atccggaatttcgtatggca atgaaagacg gtgagctggt gatatgggat 1440 agtgttcacc cttgttacaccgttttccat gagcaaactg aaacgttttc atcgctctgg 1500 agtgaatacc acgacgatttccggcagttt ctacacatat attcgcaaga tgtggcgtgt 1560 tacggtgaaa acctggcctatttccctaaa gggtttattg agaatatgtt tttcgtctca 1620 gccaatccct gggtgagtttcaccagtttt gatttaaacg tggccaatat ggacaacttc 1680 ttcgcccccg ttttcaccatgggcaaatat tatacgcaag gcgacaaggt gctgatgccg 1740 ctggcgattc aggttcatcatgccgtctgt gatggcttcc atgtcggcag aatgcttaat 1800 gaattacaac agtactgcgatgagtggcag ggcggggcgt aattttttta aggcagttat 1860 tggtgccctt aaacgcctggtgctacgcct gaataagtga taataagcgg atgaatggca 1920 gaaattcagc ttggcccagtgccaagctcc aatacgcaaa ccgcctctcc ccgcgcgttg 1980 gccgattcat taatgcagctggcacgacag gtttcccgac tggaaagcgg gcagtgagcg 2040 caacgcaatt aatgtgagttagctcactca ttaggcaccc caggctttac actttatgct 2100 tccggctcgt atgttgtgtggaattgtgag cggataacaa tttcacacag gaaaacattg 2160 accatgatta cgccgaattcgagctcggta cctcgagatc tgcagcccgg gatctccgac 2220 atcataacgg ttctggcaaatattctgaaa tgagctgttg acaattaatc atccggctcg 2280 tataatgtgt ggaattgtgagcggataaca atttcacaca ggaaacagac catggaattc 2340 ttgaatatat ggagtaagcaatgagttata ctgtcggtac ctatttagcg gagcggcttg 2400 tccagattgg tctcaagcatcacttcgcag tcgcgggcga ctacaacctc gtccttcttg 2460 acaacctgct tttgaacaaaaacatggagc aggtttattg ctgtaacgaa ctgaactgcg 2520 gtttcagtgc agaaggttatgctcgtgcca aaggcgcagc agcagccgtc gttacctaca 2580 gcgttggtgc gcattccgcattcgatgcta tcggtggcgc ctatgcagaa aaccttccgg 2640 ttatcctgat ctccggtgctccgaacaaca acgaccacgc tgctggtcat gtgttgcatc 2700 atgctcttgg caaaaccgactatcactatc agttggaaat ggccaagaac atcacggccg 2760 ccgctgaagc gatttacaccccggaagaag ctccggctaa aatcgatcac gtgattaaaa 2820 ctgctctcgc gaagaagaagccggtttatc tcgaaatcgc ttgcaacatt gcttccatgc 2880 cctgcgccgc tcctggaccggcaagtgcat tgttcaatga cgaagccagc gacgaagcat 2940 ccttgaatgc agcggttgacgaaaccctga aattcatcgc caaccgcgac aaagttgccg 3000 tcctcgtcgg cagcaagctgcgcgctgctg gtgctgaaga agctgctgtt aaattcaccg 3060 acgctttggg cggtgcagtggctactatgg ctgctgccaa gagcttcttc ccagaagaaa 3120 atccgcatta cattggtacctcatggggcg aagtcagcta tccgggcgtt gaaaagacga 3180 tgaaagaagc cgatgcggttatcgctctgg ctcctgtctt caacgactac tccaccactg 3240 gttggacgga tatccctgatcctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg 3300 tcagacgcat tcgcttccccagcgttcatc tgaaagacta tctgacccgt ttggctcaga 3360 aagtttccaa gaaaaccggttctttggact tcttcaaatc cctcaatgca ggtgaactga 3420 agaaagccgc tccggctgatccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg 3480 tcgaagctct tctgaccccgaacacgacgg ttattgctga aaccggtgac tcttggttca 3540 atgctcagcg catgaagctcccgaacggtg ctcgcgttga atatgaaatg cagtggggtc 3600 acattggttg gtccgttcctgccgccttcg gttatgccgt cggtgctccg gaacgtcgca 3660 acatcctcat ggttggtgatggttccttcc agctgacggc tcaggaagtt gctcagatgg 3720 ttcgcctgaa actgccggttatcatcttct tgatcaataa ctatggttac accatcgaag 3780 ttatgatcca tgatggtccgtacaacaaca tcaagaactg ggattatgcc ggtctgatgg 3840 aagtgttcaa cggtaacggtggttatgaca gcggtgctgc taaaggcctg aaggctaaaa 3900 ccggtggcga actggcagaagctatcaagg ttgctctggc aaacaccgac ggcccaaccc 3960 tgatcgaatg cttcatcggtcgtgaagact gcactgaaga attggtcaaa tggggtaagc 4020 gcgttgctgc cgccaacagccgtaagcctg ttaacaagct cctctagttt gagctcggta 4080 cccggggatc tccgacatcataacggttct ggcaaatatt ctgaaatgag ctgttgacaa 4140 ttaatcatcc ggctcgtataatgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 4200 acagaccatg gaattcgagctctgtagggt gaggttatag ctatggcttc ttcaactttt 4260 tatattcctt tcgtcaacgaaatgggcgaa ggttcgcttg aaaaagcaat caaggatctt 4320 aacggcagcg gctttaaaaatgcgctgatc gtttctgatg ctttcatgaa caaatccggt 4380 gttgtgaagc aggttgctgacctgttgaaa gcacagggta ttaattctgc tgtttatgat 4440 ggcgttatgc cgaacccgactgttaccgca gttctggaag gccttaagat cctgaaggat 4500 aacaattcag acttcgtcatctccctcggt ggtggttctc cccatgactg cgccaaagcc 4560 atcgctctgg tcgcaaccaatggtggtgaa gtcaaagact acgaaggtat cgacaaatct 4620 aagaaacctg ccctgcctttgatgtcaatc aacacgacgg ctggtacggc ttctgaaatg 4680 acgcgtttct gcatcatcactgatgaagtc cgtcacgtta agatggccat tgttgaccgt 4740 cacgttaccc cgatggtttccgtcaacgat cctctgttga tggttggtat gccaaaaggc 4800 ctgaccgccg ccaccggtatggatgctctg acccacgcat ttgaagctta ttcttcaacg 4860 gcagctactc cgatcaccgatgcttgcgcc ttgaaggctg cgtccatgat cgctaagaat 4920 ctgaagaccg cttgcgacaacggtaaggat atgccagctc gtgaagctat ggcttatgcc 4980 caattcctcg ctggtatggccttcaacaac gcttcgcttg gttatgtcca tgctatggct 5040 caccagttgg gcggctactacaacctgccg catggtgtct gcaacgctgt tctgcttccg 5100 catgttctgg cttataacgcctctgtcgtt gctggtcgtc tgaaagacgt tggtgttgct 5160 atgggtctcg atatcgccaatctcggtgat aaagaaggcg cagaagccac cattcaggct 5220 gttcgcgatc tggctgcttccattggtatt ccagcaaatc tgaccgagct gggtgctaag 5280 aaagaagatg tgccgcttcttgctgaccac gctctgaaag atgcttgtgc tctgaccaac 5340 ccgcgtcagg gtgatcagaaagaagttgaa gaactcttcc tgagcgcttt ctaatttcaa 5400 aacaggaaaa cggttttccgtcctgtcttg aggtacccgg ggatcctcta gagtcgacca 5460 acgtcaacaa ccacccccgcagcgttaagt tgccccgcca acagaaagga aaccaacacg 5520 aaacaacaac acaaaaaggtttcacagaaa aaagcgtatg cgctaacgta tgccccgcag 5580 cacggcaaaa gcgcgttaagccctagccca gccgcgcgta ggtattactc atgcccacta 5640 tggtgtgcac actgcccactacggtgtgca atctattcac gatgccaccc ccagatacag 5700 tgaagccccg ccaatccgaactagatcaga tcaacggggc aacccattgt ccccagcttt 5760 gattaggagc caggcacataacagcatgac agttccattc ctgatgaaat cagccattgt 5820 caacaacaag acccatcatagtttgccccc gcgacattga ccataaattc atcgcacaaa 5880 atatcgaacg gggtttatgccgcttttagt gggtgcgaag aatagtctgc tcattacccg 5940 cgaacaccgc cgcattcagatcacgcttag tagcgtcccc atgagtaggc agaaccgcgt 6000 ccaagtccac atcatccataacgatcatgc acggggtgga atccacaccc agacttgcca 6060 gcacctcatt agcgacacgttgcgcagcgg ccacgtcctt agccttatcc acgcaatcta 6120 gaacgtactg cctaaccgcgaaatcagact gaatcagttt ccaatcatcg ggcttcacca 6180 aagcaacagc aacgcgggttgattcgaccc gttccggtgc ttccagaccg gcgagcttgt 6240 acagttcttc ttccatttcacgacgtacat cagcgtctat gtaatcaatg cccaaagcac 6300 gcttagcccc acgtgaccaggacgaacgca ggtttttaga accaacctca tactcacgcc 6360 accgagccac caaaacagcgtccatatcct cgccggcgtc gctttgatcg gccaacatat 6420 ccaacatctg aaacggcgtgtacgacccct tagacgcggt tttagtagcg gagccagtca 6480 gttcctgaga catgcccttagcgaggtagg ttgccatttt cgcagcgtct ccaccccagg 6540 tagacacctg atcaagtttgaccccgtgct cacgcagtgg cgcgtccata ccggccttaa 6600 ccacaccagc agaccagcgggaaaacatgg aatcctcaaa cgccttgagt tcatcgtcag 6660 acagtggacg atccaagaacaacagcatgt gcggtgcaag tgccaaccgt tcgcccaaga 6720 gtctgtgacc tcatagtcactataggtgtg ctccaccccg taccgtgcac gttctttctt 6780 ccactgagat gttttcaccatcgaagagta cgcagtctta atacccagct ctcaacctgc 6840 gcaaatgact gtgagcggttgtgtcgaaca gtgcccacaa acatcatgag cgcgccaccc 6900 gccgccaagt gattcttagtagcaatagcc agctcaatgc ggcgttcgcc catgacttcc 6960 aattcagcca gaggtgacccccagcgagag tgagagtttt gcagaccctc aaactgcgaa 7020 cgaccgttag acgaccaggacaccgcaaca gcttcgtccc tgcgccacct atggcacccc 7080 gccagagcct tactattggtgatcttgtac atgacgtttt gcctacgcca cgccctagcg 7140 cgagtgacct tagaaccctcattgacctgc ggttccttag aggtgttcac ttctatttca 7200 gtgttaccta gacccgatgttgtgcggggt tgcgcagtgc gagtttgtgc gggtgttgtg 7260 cccgttgtct tagctagtgctatggttgtc aattgaaacc ccttcgggtt atgtggcccc 7320 cgtgcatatg agttggtagctcgcacgggg gtttgtcttg tctagggaac tattaatttt 7380 tagtggtgtt tggtggccgcctagcttggc tatgcgtgcc agcttacccg tactcaatgt 7440 taaagatttg catcgacatgggagggttac gtgtccgata cctagggggg gtatccgcga 7500 ctaggtgccc cggtgctcactgtctgtacc gcgcaagccc cacaccccgc atggaccagg 7560 tcgtccgccc cctgcacccccagcaatctg catgtacatg ttttacacat tagcacgaca 7620 tgactgcatg tgcatgcactgcatgcagac taggtaaata tgagtatgta cgactagtaa 7680 caggagcact gcacataatgaatgagttgc aggacaatgt ttgctacgca tgcgcatgac 7740 atatcgcagg aaagctactagagtcttaaa gcatggcaac caaggcacag ctagaacagc 7800 aactacaaga agctcaacaggcactacagg cgcagcaagc gcaggcacaa gccaccatcg 7860 aagcactaga agcgcaggcaaaggctaagc ccgtcgtggt caccgcacgc gttcctttgg 7920 cactacgtga ggacatgaagcgcgcaggca tgcagaacgg tgaaaacctc caagagttca 7980 tgatcgccgc gtttaccgagcggctagaaa agctcaccac caccgacaac gaggaaaaca 8040 atgtctaacc cactagttctctttgcccac cgtgacccgg taaatgacgt gacgttcgag 8100 tgcattgagc acgccacctacgacacactt tcacacgcta aagaccagat caccgcccaa 8160 atgcaagccc tagacgaagaagccgcccta ctgccctaat gggtgtttca tgggtgtttc 8220 cctagtgttt catggtgttttcacctaagc tagggaattg cgcgagaagt cctccgaaca 8280 aaatcagcaa cccccggaaccacacagttc acgggggttc ttctatgcca gaaatcagaa 8340 aggggaacca gtgaacgaccccgaatattg gatcacagcg cagcaggtcg ccgcccgcgt 8400 agctctcacc ccggccaccattaaaaagtg ggcaaacgag ggaaaaatca ccgcatacaa 8460 gatcggcaag tccgtccgattcaaagcatc agacgtagac 8500

1. A process for producing ethanol using a coryneform bacterium, whichhas been transformed by a DNA containing a gene expressing pyruvatedecarboxylase activity and, if desired, a gene expressing alcoholdehydrogenase activity under a regulatory sequence allowing for theexpression, characterized in that ethanol is prepared under conditionswherein the transformed coryneform bacterium does not substantiallyproliferate.
 2. An expression vector which is used for a process as setforth in claim 1 to transform a coryneform bacterium, into which a DNAcontaining a gene expressing pyruvate decarboxylase activity and, ifdesired, a gene expressing alcohol dehydrogenase activity isincorporated under a regulatory sequence allowing for the expression. 3.An expression vector as set forth in claim 2 wherein the vector isplasmid pKP1-PDC-ADH.
 4. A coryneform bacterium transformed with anexpression vector as set forth in claim 2 or
 3. 5. An coryneformbacterium as set forth in claim 4 wherein the bacterium isCorynebacterium glutamicum pKP1-PDC-ADH/13032.
 6. An coryneformbacterium as set forth in claim 4 wherein the bacterium isBrevibacterium lactofermentum pKP1-PDC-ADH/13869.